Congenital kidney disease occurs at a frequency of 1 in 1000 and causes significant morbidity due to renal agenesis, dysplasia and collecting system defects. Molecular genetic approaches have revealed several genes required for kidney formation. One of these is Eyes absent 1 or Eyal, which encodes a protein containing distinct transcriptional activation and protein-protein interaction domains. Haploinsufficiency for human EYA 1 results in Branchio-Oto-Renal (BOR) syndrome. Similar to individuals with BOR syndrome, Eyal heterozygous mice also exhibit renal hypoplasia and agenesis with reduced penetrance. Even more dramatically, Eyal homozygous mice manifest a fully penetrant bilateral renal agenesis phenotype due to defective ureteric bud outgrowth. Eyal is expressed in metanephric mesenchyme but not in ureteric bud. We therefore hypothesize that Eyal regulates mesenchymal factor(s) that direct ureteric bud outgrowth. Indeed, expression of GDNF, one mesenchymal factor capable of directing ureteric bud outgrowth, is undetectable in Eyal-deficient metanephric mesenchyme. In this application, we seek to resolve the Eyal-dependent genetic regulatory hierarchy controlling ureteric bud outgrowth. In Aim 1, we will use Eyal gene dosage experiments and marker studies to clarify the basis for the Eyal hetero- and homozygous phenotypes. Embryonic kidney explant cultures will be used to test whether GDNF or other mesenchymal factors can rescue ureteric bud outgrowth in Eyal homozygotes. In addition, we will use other mouse kidney mutants lacking Six1, Wtl, c-ret, Gdnf, ld (Fmnl) and Pax2 to assay dependencies of gene expression in metanephric mesenchyme and Wolffian duct. In Aim 2, we will identify proteins which Eyal interacts with in kidney development by testing known proteins, by conducting a yeast two hybrid screen, and by analyzing proteins that co-immunoprecipitate with Eyal from renal and! mesenchymal-derived cell lines. Lastly, in Aim 3, we will seek to develop a molecular model for Eyal function in nephrogenesis by testing the hypothesis that an Eyal complex can activate the Gdnf promoter, or that it can regulate the expression of other Eyal downstream target genes.